Generation of transgenic zebrafish to study electrical synaptic transmission

描述（由申请人提供）：间隙连接（GJ）介导的电突触传递被认为是神经元间通讯的基本形式。它对哺乳动物中枢神经系统不同区域的重要功能过程有重要贡献，并与各种神经系统疾病有关。电突触的可塑性通过重构电耦合神经元的网络来实现重要功能，其破坏可能导致神经功能障碍。与化学突触相反，关于调节电突触强度的分子机制知之甚少。这项建议的重点是了解机制的基础上的塑料变化GJ通信中观察到的混合，电气和化学，突触耦合初级听觉传入的硬骨鱼Mauthner（M-）细胞，在GJ的形成由鱼类同源的广泛的哺乳动物GJ蛋白连接蛋白36（Cx 36），并在那里有可能分析细胞和亚细胞机制在体内。我们在金鱼的研究表明，这两个组件的混合突触反应进行活动依赖性增强各自的优势。值得注意的是，我们最近的研究结果表明，调节营业额和功能GJ通道的数量的因素可能构成电传输的强度的主要决定因素。我们建议在这里调查的贡献，作为一种可能的机制，调节电传输的强度贩运的GJ通道。为了这个目的，我们将采取这种独特的模型混合突触到一个新的水平的分析，通过研究它们的特性在斑马鱼幼虫，其透明度将有可能跟踪活细胞内的单个分子，在体内。支持这种可能性，我们的研究结果表明，混合突触在斑马鱼幼虫的分子和功能类似的成年金鱼。该提案有两个目标：目的1是产生转基因斑马鱼，其中神经元的间隙连接蛋白与荧光蛋白标记，和目的2是调查的营业额荧光标记的间隙连接通道在体内和它的属性的条件下，触发可塑性。斑马鱼幼虫的顺从性，以图像的荧光标记的GJ通道在体内的运动应允许监测的活动突触进行可塑性提供了一个前所未有的窗口，这种传输方式的分析，详细的分子机制可以结合电生理学和活成像与强大的遗传操作进行调查。因此，这种斑马鱼模型的发展将提供一个新的强大的工具来研究Cx 36介导的突触（在哺乳动物中普遍存在）的分子方面，这可能导致识别用于治疗各种神经系统疾病的新的治疗机会。

DESCRIPTION (provided by applicant): Gap junction (GJ) mediated electrical synaptic transmission is considered an essential form of interneuronal communication. It critically contributes to important functional processes in diverse regions of the mammalian CNS and has been linked to a variety of neurological conditions. Plasticity of electrical synapses underlies important functions by reconfiguring networks of electrically coupled neurons, whose disruption might contribute to neurological dysfunction. In contrast to chemical synapses, less is known regarding the molecular mechanisms that regulate the strength of electrical synapses. This proposal focuses on understanding mechanisms underlying plastic changes in GJ communication observed at mixed, electrical and chemical, synapses that couple primary auditory afferents to the teleost Mauthner (M-) cells, at which GJs are formed by fish homologs of the widespread mammalian GJ protein connexin36 (Cx36) and where it is possible to analyze cellular and sub-cellular mechanisms in-vivo. Our studies in goldfish show that both components of the mixed synaptic response undergo activity-dependent potentiation of their respective strengths. Remarkably, our recent findings indicate that factors regulating the turnover and number of functional GJ channels might constitute major determinants of the strength of electrical transmission. We propose here to investigate the contribution of trafficking of GJ channels as a possible mechanism for regulating the strength of electrical transmission. For this purpose, we will take this unique model mixed synapse to a new level of analysis by investigating their properties in larval zebrafish, whose transparency will make it possible to track individual molecules within living cells, in vivo. Supporting this possibility, our preliminay results indicate that mixed synapses in larval zebrafish are molecularly and functionally analogous to those of adult goldfish. The proposal has two aims: Aim 1 is to generate transgenic zebrafish in which neuronal gap junction proteins are tagged with fluorescent proteins, and Aim 2 is to investigate the turnover of fluorescently tagged gap junction channels in-vivo and its properties under conditions that trigger plasticity. The amenability of zebrafish larvae to image the movement of fluorescently tagged GJ channels in-vivo should permit the monitoring of active synapses undergoing plasticity providing an unprecedented window for the analysis of this modality of transmission at which detailed molecular mechanisms could be investigated combining electrophysiology and live imaging with powerful genetic manipulations. Thus, the development of this zebrafish model will provide a new powerful tool to study molecular aspects of Cx36-mediated synapses (prevalent in mammals) that could lead to the identification of novel therapeutic opportunities for the treatment of various neurological conditions.